CN101805716B - Antagonistic bacterium for controlling bacterial wilt of capsicum and microorganism organic fertilizer thereof - Google Patents
Antagonistic bacterium for controlling bacterial wilt of capsicum and microorganism organic fertilizer thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
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Abstract
The invention relates to an antagonistic bacterium for controlling the bacterial wilt of capsicum and a microorganism organic fertilizer thereof, belonging to an agricultural intensified production technology. An antagonistic bacterium (bacillus subtilis a45) which plays an obvious antagonism role in pathogenic bacteria of the bacterial wilt of the capsicum is obtained by separation, and the disease-resistant microorganism organic fertilizer is prepared from the antagonistic bacterium and organic compost; and the fertilizer contains more than 2.0*109 a45 per gram, the total nitrogen content is 33.1g kg-1, the total phosphorus content is 13.3g kg-1 and the total potassium content is 21.3g kg-1. Tests prove that the antagonistic bacterium can be rapidly bred after the microorganism organic fertilizer is applied to soil and forms a preponderant population in soil; and the preventing and treating rate on the bacterial wilt of the capsicum is as high as 70 percent.
Description
One, technical field
The present invention relates to prevent and treat the antagonism bacterium of pepper ralstonia solanacearum and, belong to the intensive agriculture production technical field, be exclusively used in and overcome and eliminate the pepper ralstonia solanacearum problem with the microorganism organic fertilizer that it is made.
Two, background technology
Pepper ralstonia solanacearum is a kind of soil-borne disease that is caused by the withered Raul Salmonella of green grass or young crops (Ralstonia solanacearum), in case take place, causes the capsicum death of 20%-30% usually, when serious up to more than 50%, even total crop failure.At present, the control of pepper ralstonia solanacearum is mainly contained diverse ways such as shift of crops, chemical prevention and breeding resistant variety, but prevention effect is lower.Development along with green agriculture, the control emphasis of pepper ralstonia solanacearum is turned to integrated control based on biological control gradually, screening efficient antagonistic strain is the core of carrying out biological control, simultaneously antagonistic strain and composite being developed to of fertilizer can either more and more be subject to people's attention by the disease-resistant microorganism organic fertilizer that can improve Soil structure again.
Three, summary of the invention
1. technical problem
The objective of the invention is to develop a kind of antagonistic microbe organic fertilizer that can prevent and treat pepper ralstonia solanacearum, prevent and treat efficient and reach 70%, make a large amount of capsicum continuous cropping obstacle soil obtain biological restoration, guarantee agriculture the shaping up that intensify.
2. technical scheme
Be used to prevent and treat the antagonism bacterium a45 of pepper ralstonia solanacearum, belong to subtilis (Bacillus subtilis), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 30th, 2009, culture presevation number is CGMCC NO.3480, and main biological characteristics is; Gram-positive G
+, cell shape is shaft-like, gives birth in the gemma or nearly middle giving birth to, and ellipse, sporangiocyst does not expand, and does not have parasporal crystal; Glucose fermentation, fructose, pectinose, seminose, N.F,USP MANNITOL produce acid, energy hydrolyzed starch and liquefy gelatin, catalase, Citrate trianion utilization, nitrate reduction reaction, V-P (Voges-Proskauer) reaction are all positive, and clark and Lubsreaction, malonate utilization, tartrate utilization are all negative; On the LB flat board 30 ℃ cultivate 24h after, the bacterium colony periphery is irregular, and is opaque, the tool mobility.
The microorganism organic fertilizer of the control pepper ralstonia solanacearum of being produced with above-mentioned antagonism bacterium a45 is characterized in that: contain 2.0 * 10 in this fertilizer
9Above antagonism bacterium a45, the total nitrogen content of individual/g is 33.1g kg
-1, content of tatal phosphorus is 13.3g kg
-1, full potassium content is 21.3g kg
-1
The production method of the microorganism organic fertilizer of above-mentioned control pepper ralstonia solanacearum comprises:
1) bacterial strain a45 is inoculated into carries out liquid fermenting production in the PDA nutrient solution, the condition of its fermentative production is: initial pH scope is 7.0-7.2,30 ℃ of culture temperature, 170 rev/mins, the fermentation middle and later periods forms gemma, and the a45 of fermentation antimicrobial fluid short of money contains bacterium or gemma amount 〉=5 * 10
9Individual/ml; Used PDA nutrient solution compound method is, is example with preparation 1L substratum: be cut into small pieces to be put in the water after peeling with the 200g potato and boil, boil 30min after the boiling, add the 20g common sucrose after filtering in the filtrate, be settled to 1000ml, the pH value transfers to 7.2-7.4,121 ℃ of sterilization 20min.
2) preparation of fertilizer: be mixed and made at 3: 3: 4 with volume ratio with the pig manure that becomes thoroughly decomposed, the cow dung compost of becoming thoroughly decomposed, amino acid fertilizer; The used pig manure that becomes thoroughly decomposed, cow dung compost germination index are more than 98%, quality of organic matter ratio content 〉=35%, and the organonitrogen quality is 1.2-2% than content, water content 25-30%.
3) a45 antimicrobial fluid short of money is inoculated into step 2 in the ratio of 20% (V/W)) carry out solid fermentation in the described fertilizer, leavening temperature is 30-50 ℃, turning every day is 1 time in the fermenting process, fermentation period 5-7 days, makes the antagonism bacterial content reach 2.0 * 10
9Individual/as more than the g, under temperature is no more than 60 ℃ condition its water content to be evaporated to below 30% at last, packing is dispatched from the factory and is the microorganism organic fertilizer of control pepper ralstonia solanacearum.
Described microorganism organic fertilizer can be exclusively used in the control pepper ralstonia solanacearum.
3. beneficial effect
The present invention relates to a kind of microorganism organic fertilizer and production method thereof that can overcome or eliminate the continuous cropping pepper ralstonia solanacearum, utilize pig manure, cow dung compost and amino acid fertilizer mixture, make microorganism organic fertilizer again with after antimicrobial fluid short of money is mixed, its product is compared with product in the market has following advantage:
1) contain the efficient bacterial strain of specificity (subtilis a45) that suppresses the growth of pepper ralstonia solanacearum pathogenetic bacteria in this fertilizer product, it suppresses the effect highly significant.Test-results shows, contain use this product on the soil of pepper ralstonia solanacearum after, the biological control rate of pepper ralstonia solanacearum is up to more than 70%.
2) this fertilizer is fertilizer, contains abundant organic matter, and major part is amino acid and micromolecule polypeptide.The abundant nutrition that contains in the organic fertilizer also provides the condition of growth and breeding for antagonism bacterium wherein, make its in soil, survive under and form dominant population, thereby the performance prophylaxis effect.
3) owing to be biological bacterial strain preparation, there are not a series of problems that use brought fully because of chemical pesticide, thereby help the nuisanceless production of capsicum, the peasant can or reduce the consumption of the chemical pesticide of preventing and treating bacterial wilt, this not only can be the peasant and reduces expenses, and helps the outlet of agricultural-food.Simultaneously, this microorganism organic fertilizer increases production function in addition, can be the peasant and increases income.
Four, description of drawings
Fig. 1 uses the influence to capsicum root system pathogenic bacteria of antagonism bacterium a45 and microbial organic fertilizer
Five, embodiment
(1) separation of bacterial strain and evaluation
With the blue or green withered Raul Salmonella (Ralstonia solanacearum) of the pepper ralstonia solanacearum pathogenic bacteria (DSMZ (DSMZ) of Germany, Ralstoni a solanacearum (DSM No.9544), the screening of pepper ralstonia solanacearum antagonistic strain and the mensuration of field efficacy, " Chinese biological control " calendar year 2001, the 17th volume, the 03rd phase, Guo Jianhua etc.) for separating the antagonism bacterium in the healthy pepper plant rhizosphere soil of indicator from come from pepper ralstonia solanacearum morbidity field piece.Select efficient antagonistic strain a45 by the pot experiment finishing screen again, identify to belong to and plant, bacterial classification is-70 ℃ of preservations in 30% glycerine.Antagonism bacterium a45 belongs to subtilis (Bacillus subtilis), and main biological characteristics is: Gram-positive G
+, cell shape is shaft-like, gives birth in the gemma or nearly middle giving birth to, and ellipse, sporangiocyst does not expand, and does not have parasporal crystal; Glucose fermentation, fructose, pectinose, seminose, N.F,USP MANNITOL produce acid, energy hydrolyzed starch and liquefy gelatin, catalase, Citrate trianion utilization, nitrate reduction reaction, V-P (Voges-Proskauer) reaction are all positive, and clark and Lubsreaction, malonate utilization, tartrate utilization are all negative; On the LB flat board 30 ℃ cultivate 24h after, the bacterium colony periphery is irregular, and is opaque, has mobility.
(2) microbial inoculum production
1) inoculation is carried out liquid fermenting production in the PDA nutrient solution, the condition of its fermentative production is: initial pH scope is 7.0-7.2,30 ℃ of culture temperature, 170 rev/mins, the fermentation middle and later periods forms gemma, obtains a45 antimicrobial fluid short of money (LC) and contains bacterium or gemma amount 〉=5 * 10
9Individual/ml;
Used PDA nutrient solution compound method is, is example with preparation 1L substratum: be cut into small pieces to be put in the water after peeling with the 200g potato and boil, boil 30min after the boiling, add the 20g common sucrose after filtering in the filtrate, be settled to 1000ml, the pH value transfers to 7.2-7.4,121 ℃ of sterilization 20min.
2) preparation of fertilizer (OF): with pig manure, cow dung compost and the amino acid fertilizer (Jiangsu Xintiandi amino acid fertilizer Co.ltd of becoming thoroughly decomposed, fertilizer title: amino acid fertilizer, No. 0180, the accurate word of registration card number: Su Nongfei (2008), fertilizer specification: N+P
2O
5+ K
2O 〉=6% organic matter 〉=30% amino acid 〉=5%) with volume ratio 3: 3; 4 are mixed and made into.The used pig manure that becomes thoroughly decomposed, cow dung compost germination index are more than 98%, quality of organic matter ratio content 〉=35%, and the organonitrogen quality is 1.2-2% than content, water content 25-30%.
3) a45 antimicrobial fluid short of money is inoculated into step 2 in the ratio of 20% (V/W)) carry out the secondary solid fermentation in the described fertilizer, leavening temperature is 30-50 ℃, and turning every day is 1 time in the fermenting process, and fermentation fermentation period 5-7 days makes the antagonism bacterial content reach 2.0 * 10
9Individual/as more than the g, to obtain the solid fungicide of antagonism bacterium a45.At last the water content that is no more than under 60 ℃ the condition microbial organic fertilizer in temperature is evaporated to below 30%, obtains the microorganism organic fertilizer (BOF) of control pepper ralstonia solanacearum.
Microbial organic fertilizer (BOF) (microbial organic fertilizer special of control pepper ralstonia solanacearum) with above-mentioned antagonism bacterium is produced contains 2.0 * 10 in this fertilizer
9The antagonism bacterium a45 of individual/g, total nitrogen content are 33.1g kg
-1, content of tatal phosphorus is 13.3g kg
-1, full potassium content is 21.3g kg
-1
(3) greenhouse pot culture test
Be transplanted in the basin alms bowl that 5kg soil is housed behind 4～5 true leaves of growing seedlings available from Vegetable ﹠ Flower Inst., Chinese Academy of Agriculture Science for green pepper No. 4 in the examination capsicum variety.The basin alms bowl is provided with 5 processing:
1) healthy soil (CK
1);
2) healthy soil+pepper ralstonia solanacearum pathogenic bacteria (CK
2);
3) healthy soil+pepper ralstonia solanacearum pathogenic bacteria+fertilizer (CK
2+ OF);
4) healthy soil+pepper ralstonia solanacearum pathogenic bacteria+a45 antimicrobial fluid (CK short of money
2+ LC);
5) healthy soil+pepper ralstonia solanacearum pathogenic bacteria+microbial organic fertilizer (CK
2+ BOF).
Three district's groups are established in every kind of processing, and each district organizes 12 repetitions.Wherein fertilizer (OF) and microbial organic fertilizer amount of application (BOF) are 5 ‰, do not add processing nitrogen, phosphorus, the potash fertilizer polishing nutrient of fertilizer.According to being provided with pathogenic bacteria and antagonism bacterium are inoculated into respectively in the soil, pathogenic bacteria content is 9.6 * 10 in the soil of inoculation pathogenic bacteria
6Individual/g soil, inoculate that antimicrobial content short of money is 1.0 * 10 in the antimicrobial soil short of money
7Individual/g soil, specific practice is: adopting bacteria containing amount is 2.0 * 10
9The microbial organic fertilizer of individual/g applies fertilizer according to 5 ‰ rates of fertilizer application (that is: using 25g in the 5Kg soil), makes that antimicrobial content short of money is 1.0 * 10 in the soil
7Individual.
Chili growth after 15 days every day investigate bacterial wilt a situation arises, calculate disease index and prevention effect (disease index=∑ (diseased plant numbers at different levels * corresponding progression)/investigation total plant number * 100; Prevention effect=(check plot disease index-treatment zone disease index)/check plot disease index * 100; Disease severity grade scale: 0 grade: no symptom; I level: 1/4 blade morbidity; II level: 1/2 blade morbidity; III level: whole strain death).The result shows, uses antagonism bacteria liquid microbial inoculum and microbial organic fertilizer the biological control rate of pepper ralstonia solanacearum is respectively 69.58% and 76.09% (table 1).Can also significantly reduce simultaneously the quantity (Fig. 1) of rhizosphere pathogenic bacteria, the quantity that delays a45 descends.
Test-results shows, use microbial organic fertilizer and not only can play the control pepper ralstonia solanacearum, significantly reduce the effect of the former bacterium quantity of bacterial wilt in the soil, also promote the variation of capsicum rhizosphere microflora, beneficial bacteria, actinomycetes and fungal colonization all significantly increase (table 2).
Table 1 antagonism bacterium a45 and with the fertilizer composite application to the control pepper ralstonia solanacearum effect
| Handle | Disease index (%) | Prevention effect (%) |
| Healthy soil (CK 1) | - | - |
| Healthy soil+pepper ralstonia solanacearum pathogenic bacteria (CK 2) | 56.80±5.76a | - |
| Healthy soil+pepper ralstonia solanacearum pathogenic bacteria+fertilizer (CK 2+OF) | 43.21±6.58b | 23.93±4.55b |
| Healthy soil+pepper ralstonia solanacearum pathogenic bacteria+a45 antimicrobial fluid (CK short of money 2+LC) | 17.28±3.29c | 69.58±3.57a |
| Healthy soil+pepper ralstonia solanacearum pathogenic bacteria+microbial organic fertilizer (CK 2+BOF) | 13.58±3.29c | 76.09±3.78a |
Table 2 is used microbial organic fertilizer to microbiotic influence in the capsicum rhizosphere soil
| Handle | Bacterium (* 10 9CFU/g soil) | Actinomycetes (* 10 6CFU/g soil) | Fungi (* 10 4CFU/g soil) |
| CK 1 CK 2 CK 2+OF CK 2+LC CK 2+BOF | 0.42±0.02b 0.52±0.11b 1.30±0.37a 1.60±0.05a 1.58±0.26a | 4.82±0.52cd 4.07±0.94d 6.63±0bc 7.69±1.02b 11.30±0a | 2.19±0.19b 5.94±0.74b 9.00±0b 19.70±3.65a 20.20±3.50a |
Claims (6)
1. be used to prevent and treat the antagonism bacterium a45 of pepper ralstonia solanacearum, belong to subtilis (Bacillus subtilis), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 30th, 2009, culture presevation number is CGMCC NO.3480, and main biological characteristics is: Gram-positive G
+, cell shape is shaft-like, gives birth in the gemma or nearly middle giving birth to, and ellipse, sporangiocyst does not expand, and does not have parasporal crystal; Glucose fermentation, fructose, pectinose, seminose, N.F,USP MANNITOL produce acid, energy hydrolyzed starch and liquefy gelatin, catalase, Citrate trianion utilization, nitrate reduction reaction, V-P reaction are all positive, and clark and Lubsreaction, malonate utilization, tartrate utilization are all negative; On the LB flat board 30 ℃ cultivate 24h after, the bacterium colony periphery is irregular, and is opaque, has mobility.
2. the microorganism organic fertilizer of being produced with the antagonism bacterium a45 of the described control pepper ralstonia solanacearum of claim 1 is characterized in that: contain 2.0 * 10 in this fertilizer
9Above antagonism bacterium a45, the total nitrogen content of individual/g is 33.1g kg
-1, content of tatal phosphorus is 13.3g kg
-1, full potassium content is 21.3g kg
-1
3. the production method of the described control pepper ralstonia solanacearum of claim 2 microorganism organic fertilizer comprises:
1) the described bacterial strain a45 of claim 1 is inoculated into carries out liquid fermenting production in the PDA nutrient solution, the condition of its fermentative production is: initial pH scope is 7.0-7.2,30 ℃ of culture temperature, 170 rev/mins, the fermentation middle and later periods forms gemma, and the a45 antimicrobial fluid short of money that fermentation obtains contains bacterium or gemma amount 〉=5 * 10
9Individual/ml;
2) preparation of fertilizer: make with 3: 3: 4 mixed of volume ratio with the pig manure that becomes thoroughly decomposed, the cow dung compost of becoming thoroughly decomposed and amino acid fertilizer;
3) with a45 antimicrobial fluid short of money by volume the ratio of mass ratio 20% be inoculated into step 2) carry out solid fermentation in the described fertilizer, leavening temperature is 30-50 ℃, turning every day is 1 time in the fermenting process, fermentation period 5-7 days, makes the antagonism bacterial content reach 2.0 * 10
9Individual/as more than the g, under temperature is no more than 60 ℃ condition, water content to be evaporated to below 30% at last, the dispatch from the factory microorganism organic fertilizer of the control pepper ralstonia solanacearum that is acquisition of packing.
4. according to the production method of the described microorganism organic fertilizer of claim 3, wherein the used PDA nutrient solution of step 1) compound method is, with preparation 1L substratum is example: be cut into small pieces to be put in the water after peeling with the 200g potato and boil, boil 30min after the boiling, add the 20g common sucrose after filtering in the filtrate, be settled to 1000ml, the pH value transfers to 7.2-7.4,121 ℃ of sterilization 20min.
5. according to the production method of claim 3 or 4 described microorganism organic fertilizers, the used pig manure that becomes thoroughly decomposed, the cow dung compost germination index that becomes thoroughly decomposed are more than 98%, quality of organic matter ratio content 〉=35%, the organonitrogen quality is 1.2-2% than content, water content 25-30%.
6. the application of the described microorganism organic fertilizer of claim 2.
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| CN101948780B (en) * | 2010-09-01 | 2012-05-09 | 南京农业大学 | Antagonist bacterium for preventing and treating continuous cropping hot pepper epidemic disease and microbial organic fertilizer thereof |
| CN103074272A (en) * | 2012-12-17 | 2013-05-01 | 漳州三炬生物科技有限公司 | Bacillus methylotrophicus Sanju-04 and its application |
| CN104531588A (en) * | 2015-01-07 | 2015-04-22 | 南京农业大学 | Effective antagonistic strain for preventing and controlling continuous cropping tobacco bacterial wilt disease, microorganism organic fertilizer including effective antagonistic strain, and production method and application of effective antagonistic strain |
| CN104961538B (en) * | 2015-05-08 | 2017-11-14 | 淮阴工学院 | Recessed soil matrix immobilization complex micro organism fungicide for improveing capsicum continuous cropping obstacle and preparation method thereof |
| CN108676778B (en) * | 2018-04-20 | 2022-09-20 | 南京农业大学 | Bacteriophage for preventing and treating soil-borne bacterial wilt and application thereof |
| CN108642018B (en) * | 2018-04-26 | 2022-08-05 | 南京农业大学 | Lytic bacteriophage capable of preventing and controlling tomato bacterial wilt and application thereof |
| CN108821863A (en) * | 2018-07-12 | 2018-11-16 | 山东博华高效生态农业科技有限公司 | A kind of functional biological organic fertilizer and preparation method thereof |
| CN108586116A (en) * | 2018-08-10 | 2018-09-28 | 保康丰源肥业有限责任公司 | A kind of biological organic fertilizer and preparation method thereof of prevention pepper ralstonia solanacearum |
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